KR20100108569A - Human bone-forming cells in the treatment of inflammatory rheumatic diseases - Google Patents

Abstract

The present invention relates in particular to the novel therapeutic use of isolated bone-forming cells in the treatment of inflammatory rheumatic diseases.

Description

HUMAN BONE-FORMING CELLS IN THE TREATMENT OF INFLAMMATORY RHEUMATIC DISEASES

The present invention relates to the therapeutic application of bone-forming cells in the treatment of inflammatory rheumatic disease (IRD), in particular in the treatment of inflammation in IRD (inflammatory component of IRD).

Rheumatoid disease includes a variety of painful disorders that affect the kinetics, including joints, muscles, connective tissue, soft tissues surrounding joints and bones in particular.

Inflammatory and / or autoimmune responses contribute to the pathogenesis of many rheumatic diseases. Such conditions, commonly referred to as inflammatory rheumatic disease or IRD, include, but are not limited to arthritis, osteoarthritis, etc. of various origins.

Currently available treatments for IRD include primarily disease-modulating antirheumatic drugs (DMARDs), glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), and analgesics.

Thus there is a need for additional modalities in IRDs, in particular modalities that target the inflammatory component of IRDs.

WO 2005/089127 includes osteogenic cells in skeletal devices for regenerating osteochondral interfaces in osteoarthritis. WO 2007/093431 proposes the use of osteoblasts for the treatment of rheumatoid arthritis and osteonecrosis. These documents do not disclose the anti-inflammatory action of bone-forming cells and do not disclose the use of bone-forming cells to inhibit the inflammatory component of rheumatoid disease.

Liu et al. 2006 (J Immunol 176 (5): 2864-71) discloses immune evasion and immunomodulatory properties of osteogenic cells differentiated from mesenchymal stem cells in the context of allogeneic tissue transplantation. However, the tissue rejection mechanism in allogeneic transplantation is clearly different from the underlying inflammatory mechanism in rheumatoid disease. This is reflected in, among other things, separate groups of drugs currently used to treat these conditions. As a result, Liu et al. 2006 does not disclose any anti-inflammatory action of bone-forming cells, nor does it disclose the use of bone-forming cells to inhibit the inflammatory component of rheumatoid disease.

The inventors surprisingly show that bone-forming cells exhibit potent immunosuppression, in particular anti-inflammatory action, in addition to the expected bone regeneration action, and thus in inflammatory rheumatic disease (IRD) in a subject, more particularly in IRD in a subject. Ie it is particularly useful for treating inflammation in the inflammatory component of IRD.

Accordingly, aspects of the present invention provide the use of isolated bone-forming cells for the preparation of a medicament for the treatment of IRD as well as isolated bone-forming cells for use in treating IRD. Further disclosed is the use of isolated bone-forming cells for the preparation of a medicament for treating inflammation in the IRD (inflammatory component) as well as isolated bone-forming cells for use in treating inflammation in the IRD (inflammatory component of IRD). do.

The invention also relates to a method for preventing and / or treating IRD in a subject in need thereof, comprising administering to said subject a prophylactic or therapeutically effective amount of isolated bone-forming cells. Also disclosed are methods for preventing and / or treating inflammation in an IRD (inflammatory component of IRD) in a subject in need thereof, comprising administering to the subject in need thereof a prophylactically or therapeutically effective amount of isolated bone-forming cells. . The present invention also relates to pharmaceutical compositions comprising isolated bone-forming cells for use in treating IRD. Also disclosed is a pharmaceutical composition comprising isolated bone-forming cells for use in treating inflammation in an IRD (inflammatory component of IRD).

Also described are the use of isolated bone-forming cells for the manufacture of a medicament for treating inflammation as well as isolated bone-forming cells for use in treating inflammation. In addition, methods are described for preventing and / or treating inflammation in a subject in need thereof, comprising administering to the subject in need thereof a prophylactically or therapeutically effective amount of isolated bone-forming cells. Further described are pharmaceutical compositions comprising isolated bone-forming cells for use in treating inflammation.

The term "isolated" as used herein with respect to a cell or cell population means that the cell or cell population is removed or separated from it without forming part of an animal or human body.

The bone-forming cells may be of mammalian origin, more preferably of human origin, preferably including non-human mammalian origin. Bone-forming cells are usually obtained from or derived from a biological sample of a subject, such as a human or non-human mammalian subject (ie, a sample removed from comprising the subject and its cells).

The subject is preferably a warm blooded animal, more preferably a mammalian subject comprising a human and a non-human mammal subject, even more preferably a primate subject comprising a human and a non-human primate subject, even more preferably a human subject It includes. Thus bone-forming cells may also be of such origin.

The bone-forming cells are preferably autologous administration (ie, administered to the same subject from which the cells are obtained or derived) or allogeneic administration (ie, the subject from which the cells are obtained or derived from). Other subjects but administered to subjects of the same species). Heterologous administration of such bone-forming cells (ie, cells obtained or derived from one species of subject are administered to subjects of different species) may also be possible.

Preferably, human bone-forming cells are used for autologous or allogeneic administration to a human subject having an IRD. Self-administration is particularly preferred.

The term "bone-forming cell" as used herein generally refers to a cell that can contribute to or give rise to a cell that can contribute to the formation of bone material and / or bone matrix, in particular a) osteogenic differentiation Isolated cell that can be rough, b) is involved in osteogenic differentiation, or c) at least partially progresses along osteogenic differentiation, more preferably enumerated cells listed under any b) or c) Or cell populations. Without limitation, bone-forming cells include especially osteoprogenitors, osteoblasts, osteocytes and other cell types of the osteogenic lineage as known in the art.

Thus, those skilled in the art generally understand the scope of the term "bone-forming cell" as intended herein. Nevertheless, without limitation by further guidance, the bone-forming cells may exhibit any one or more, or all of the following features:

a) the cell comprises the expression of alkaline phosphatase (ALP), more particularly the ALP of the bone-liver-kidney type, or the expression of osteocalcin or both;

b) optionally, the cell expresses any one or more of procollagen type 1 amino-terminal propeptide (P1NP), osteonectin (ON), osteopontin (OP) and bone sialoprotein (BSP) It includes;

c) optionally, the cell comprises expression of any one or more mesenchymal markers CD105, CD73, and CD90;

d) Cells show evidence of their ability to mineralize the external environment or to synthesize calcium-containing extracellular matrix (eg, when exposed to osteogenic media; Jaiswal et al. 1997. J Cell Biochem 64: 295-312). ). Deposited into the calcium accumulation, and matrix protein within the cell is typically, for example, by the presence in the culture in the ⁴⁵ Ca ^{2 +,} washed and grown amount, since the cell or cells measures a random radioactivity deposited in the extracellular matrix (U.S. Patent No. No. 5,972,703), or Ca ^{2 +} test kit (using Sigma kit # 587) by testing the culture substrate for the mineralization, or may be measured as described in example;

e) the cell substantially differentiates into any of the cells of an adipocyte lineage (eg adipocyte) or chondrocyte lineage (eg chondrocyte), preferably either It doesn't work. The absence of differentiation into said cell lineage is defined by standard differentiation induction conditions established in the art (see, eg, Pittenger et al. 1999. Science 284: 143-7), and assays (e.g. adipocytes, if induced). Normally stained with oil red O, indicating lipid accumulation; chondrocytes can be tested using alkian blue or safranin O normally. The actual lack of propensity to adipocyte and / or chondrocyte differentiation usually results in less than 50%, or less than 30%, or less than 5%, or less than 1% test cells applied to each test. It is meant to show signs of red or chondrocyte differentiation.

In one embodiment, the bone-forming cells can exhibit all the features listed in a), d) and e) above.

A cell is said to be positive for a particular component (e.g., a marker or an enzyme), either by detection by the reverse transcriptase polymerase chain reaction to that component, or antibody-, as compared to a suitable control when the skilled person makes appropriate measurements. It will conclude the presence or evidence of a separate signal such as detectability. For methods that allow quantitative evaluation of components, signals on which positive cells differ significantly from the control on average, for example, but not limited to, at least 1.5 times higher than, for example, 2 times, signals generated by control cells At least 4 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times higher or higher.

The expression of the cell-specific markers can be determined by any suitable immunological technique known in the art, such as immune cytochemistry or affinity adsorption, Western blot analysis, FACS, ELISA, or the like, or (eg, for ALP). Any suitable biochemical assay of enzymatic activity, or any suitable technique for measuring the amount of marker mRNA, for example Northern blot, semi-quantitative or quantitative RT-PCR and the like can be detected. Sequence data for the markers listed in this disclosure are known and can be obtained from public databases such as GenBank (http://www.ncbi.nlm.nih.gov/).

Isolated bone-forming cells or cell populations for use in the present invention may be obtained or derived by any suitable method known in the art. In one embodiment the bone-forming cells or cell populations are derived by differentiation from relatively less differentiated adult progenitors or stem cells (eg mesenchymal stem cells), using their own known differentiation protocols. Can be. One non-limiting method for obtaining bone-forming osteoblasts is disclosed in WO 2007/093431 and isolated bone marrow stem cells (BMSCs) or mesenchymal stems in the presence of plasma and basic fibroblast growth factor (FGF-2). Culturing cells (MSCs). In another example, osteogenic lineage cells are described in Pittenger et al. 1999 (Science 284: 143-7) and Jaiswal et al. 1997 can be obtained by differentiating MSC in osteogenic medium. In another embodiment, bone-forming cells or cell populations can be isolated, optionally cultured, and / or expanded from a biological sample comprising said cells. For example, osteoblasts are described in Skjodt et al. 1985 (J Endocrinol 105: 391-6), can be isolated and cultured directly from the trabecular bone.

The term "inflammatory rheumatic disease" or "IRD" as used herein generally includes all rheumatic diseases involving inflammatory and / or autoimmune components, especially involving at least one inflammatory component. By way of example and not limitation, IRDs in particular include osteoarthritis (OA), psoriatic arthrosis, gout, pseudogout, and arthritis of various origins, including rheumatoid arthritis (RA), enteroarthritis, reactive arthritis, and lighter syndrome, Osteonecrosis, minor arthritis juvenile rheumatoid arthritis, Still's disease, Behcet's disease, systemic lupus erythematosus, septic arthritis and spondyloarthropathies such as ankylosing spondylitis and enteropathic spondylitis and undifferentiated spondyloarthropathy.

Thus, in one embodiment, the present disclosure provides osteoarthritis (OA), psoriatic arthrosis, gout, pseudogout, and arthritis of various origins, including rheumatoid arthritis (RA), enteroarthritis, reactive arthritis, and lighter syndrome , Osteonecrosis, minor arthritis juvenile rheumatoid arthritis, Still's disease, Behcet's disease, systemic lupus erythematosus, septic arthritis and spondyloarthropathies, including, among others, ankylosing spondylitis and enteropathic spondylitis and undifferentiated spondyloarthropathy It may be related to the IRD.

In another embodiment, the present disclosure may relate to any one IRD other than osteoarthritis (OA), osteonecrosis and rheumatoid arthritis (RA).

In another embodiment, the present disclosure is directed to psoriatic arthrosis, gout, pseudogout, and arthritis of various origins, including enteroarthritis, reactive arthritis, and Reiter syndrome, minor joint juvenile rheumatoid arthritis, Still's disease, It may be related to any one IRD selected from Behcet's disease, systemic lupus erythematosus, septic arthritis, and spondyloarthropathy including, among others, ankylosing spondylitis and enteropathic spondylitis and undifferentiated spondyloarthropathy.

The bone-forming cells may be particularly useful for treating an IRD disease (or for treating an inflammation or inflammatory component in the disease), and for bone lesion (s) such as inflammation with and without autoimmune components, and erosion or Includes all subchondral lesions. In this embodiment, more significant therapeutic improvement can be achieved by the bone-forming cells synergistically enhancing the pathology.

In another embodiment, bone-forming cells can be used to treat IRD disease (or to treat inflammation or inflammatory components in the disease), wherein the disease includes inflammation and does not include bone lesion (s). By way of example, IRD disease can be treated in patients in which an inflammatory component is present and bone lesion (s) has not yet occurred subsequently. The use of bone-forming cells in such diseases has not been shown previously, which prior to the present disclosure on the anti-inflammatory effects of bone-forming cells may be useful for any benefit in the disease from administration of bone-forming cells. Because there was no expectation.

Prevention and / or treatment of inflammation in an IRD (ie, an inflammatory component of the IRD) inhibits, reduces or reduces the level or extent of systemic and / or local inflammation in particular in the IRD, ie inhibits inflammation (inflammatory component). , Reducing or lowering. The level or extent of inflammation is known per se, for example, but not limited to, measuring symptoms of inflammation (eg, heat, tissue swelling, pain, loss of function, etc.) and / or cellular and / or inflammation. Or by measuring molecular markers (eg IL-1α, IL-1β, IL-2, IL-6, IL-8 and TNFα, such markers at local and / or systemic levels). Can be.

Isolated bone-forming cells for use in the present invention may be suitably formulated and administered as pharmaceutical compositions.

The pharmaceutical composition may further comprise pharmaceutically acceptable excipients, carriers, buffers, preservatives, stabilizers, antioxidants or other substances well known to those skilled in the art in addition to bone-forming cells as described herein. The material must be nontoxic and should not interfere with the activity of the cell. The specific nature of the carrier or other material will depend on the route of administration. For example, the composition may be in the form of a parenterally acceptable aqueous solution (pyrogen-free and having suitable pH, isotonicity and stability). For general principles in medical formulations, readers refer to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds., Cambridge University Press, 1996; And Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

Such pharmaceutical compositions may additionally contain components which ensure the viability of the cells therein. For example, the composition may comprise a buffer system (e.g., a phosphate or carbonate buffer system) suitable for obtaining a pH close to the desired pH, more typically close to neutral pH, and isotonic on cells to prevent osmotic stress. Sufficient salts may be included to ensure osmotic conditions. For example, a suitable solution for these purposes can be phosphate buffered saline (PBS), sodium chloride solution, Ringer's Injection or Lactated Ringer's Injection as known in the art. In addition, the composition may comprise a carrier protein, for example albumin, which may increase the viability of the cell.

The pharmaceutical composition may comprise additional ingredients useful for the treatment of bone wounds and defects. For example, the component may include, but is not limited to, hydroxyapatite / tricalcium phosphate particles (HA / TCP), gelatin, polylactic acid, polylactic glycolic acid, hyaluronic acid, chitosan, poly-L-lysine and collagen. For example, bone-forming cells can be bone-induced as well as combined with demineralized bone matrix (DBM) or other substrates (self-forming bone) to produce complex osteogenicity. Good results were obtained with similar methods using autologous bone marrow cells with allogeneic DBM (Connolly et al. 1995. Clin Orthop 313: 8-18).

The pharmaceutical composition may further comprise or co-administer with complementary bioactive factors or any other growth factors, such as bone morphogenic proteins (eg BMP-2 or BMP-4, BMP-7). Other potential companion ingredients include inorganic sources of calcium or phosphate that are suitable for helping bone regeneration (WO 00/07639). If desired, cellular preparations may be administered to a carrier substrate or material to provide improved tissue regeneration. For example, the material may be granular ceramic or biopolymer such as gelatin, collagen, osteonectin, fibrinogen, or osteocalcin. Porous substrates may be synthesized according to standard techniques (eg, Mikos et al., Biomaterials 14: 323, 1993; Mikos et al., Polymer 35: 1068, 1994; Cook et al., J. Biomed. Mater. Res. 35: 513 , 1997).

The pharmaceutical composition is co-administered with any therapy known in the art useful for IRD, including but not limited to disease-modulating antirheumatic drugs (DMARDs), glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs) or analgesics Or may further comprise it. Therefore, the present invention also provides a pharmaceutical composition comprising an agent selected from DMARD, glucocorticoid, NSAID and analgesic and bone-forming cells for simultaneous, sequential or individual use in IRD treatment.

Bone-forming cells may be a “prophylactically effective amount” (ie, an amount that inhibits or delays the onset of a disorder in a subject, as explored by a researcher, veterinarian, physician, or other clinician) or “therapeutically effective amount” (ie, Will be administered in an amount that elicits a biological or medical response in the subject, which may include alleviation of the symptoms of the disease or disorder to be treated, as sought by a researcher, veterinarian, physician or other clinician. Dosage or amount of bone-forming cells used (optionally used with one or more other active agents) depends on the individual case and is conventional and is adapted to the individual environment to achieve the optimum effect. Thus, this indicates the severity and nature of the disorder to be treated, and also the sex, age, weight, general health, diet, method and time of administration, and individual responsiveness, route of administration, efficacy, stability and duration of action, acute treatment Whether it is cognitive or chronic or prophylactic, or whether other active compounds are additionally administered to the bone-forming cells of the invention.

Examples include, but are not limited to, about 1 × 10 ³ to about 1 × ^{10 9} bone forming cells, or about 1 × 10 ⁴ to about 1 × ^{10 8} bone forming cells, or about 1 × 10 ⁵ to about 1 × 10 ⁷ bone forming cells, or about 1 × 10 ⁶ to 1 × ^{10 8} bone forming Doses of cells may be administered to the subject, preferably non-human mammals or human subjects, in local and / or systemic administration. Such administration may be single or repeated, or may be carried out in volume units. By way of example and not limitation, the cycle of repeated administration may be once or twice per day; Once, twice or more per week; Or once or twice per month.

The invention further includes a method of making the pharmaceutical composition, wherein the pharmaceutical composition is intended for use in treating IRD by mixing bone-forming cells as disclosed herein with the one or more additional ingredients.

Bone-forming cells or pharmaceutical compositions comprising the same can be administered or transplanted to the intended tissue site and in such a way that the functional deficient areas are reconstituted or regenerated. Administration of the composition will depend on the musculoskeletal site to be treated. For example, administration can occur by direct injection or implantation into the intraarticular cavity in the case of a joint disorder. In other circumstances, bone-forming cells or pharmaceutical compositions comprising the same can be administered systemically so that their anti-inflammatory action can occur systemically or migrate to the disease area. Therefore, in general examples, administration may be systemic, topical, intraarticular or perarticular administration, among others.

Thus, in one embodiment, the pharmaceutical cell preparation as defined above can be administered in the form of a liquid composition.

In another embodiment, bone-forming cells or cell populations can be migrated to and / or cultured in a suitable substrate to provide an implant. Substrates to which cells may be applied and cultured may be metals such as titanium, cobalt / chromium alloy or stainless steel, bioactive surfaces such as calcium phosphate, polymer surfaces such as polyethylene, and the like. Although less preferred, siliceous materials such as glass ceramics may also be used as the substrate. Although calcium phosphate is not an essential component of the substrate, metals such as titanium and calcium phosphate are most preferred. The substrate can be porous or non-porous.

For example, cells that proliferate or differentiate in a culture dish may be transferred to a three-dimensional solid support and, if necessary, induced to multiply and / or sustain the differentiation process by incubating the solid support in the liquid nutrient medium of the present invention. The cells can be transferred to the support, for example, by impregnating a three-dimensional solid support with the cell containing liquid suspension. The impregnated support obtained in this manner can be implanted in a human subject. The impregnated support can also be cultivated by soaking it in liquid culture medium before finally implanting.

The three-dimensional solid support needs to be biocompatible to be implantable in humans. It may be in any suitable form such as a cylinder, sphere, plate or part of an indefinite form. Among the materials suitable for biocompatible three-dimensional solid supports, calcium carbonate can be mentioned in particular, and in particular, mention can be made of porcelain in the form of coral skeleton, alumina, zirconia, tricalcium phosphate and / or hydroxyapatite-based porous ceramics, Restrictive coral backbones are obtained by hydrothermal exchange that allows calcium to be transformed into hydroxyapatite, or bioactive glass ceramics such as apatite- wollastonite glass ceramics, Bioglass (TM) glass.

General definition

Unless defined otherwise, all terms used in this disclosure, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the singular includes both the singular and the plural unless the context clearly dictates otherwise. By way of example, "cell" refers to one or more cells.

As used herein, the terms “comprising”, “comprises” and “consisting of” are synonymous with “comprising”, “comprises” or “comprising”, “comprising”, “inclusive” or “open”. And does not exclude additional non-cited members, elements or method steps.

Citation of numerical ranges by endpoints includes cited endpoints as well as all numerical and fractional numbers falling within the range.

As used herein, the term “about”, when referring to a measurable value such as a parameter, amount, temporal persistence, and the like, is within a range suitable for a deviation to perform in the disclosed invention, from a particular value, and from a particular value. / -10% or less, preferably +/- 5% or less, more preferably +/- 1% or less, even more preferably +/- 0.1% or less. It will also be understood that the value indicated by the modifier "about" is also specifically and preferably disclosed.

All documents cited herein are incorporated by reference in their entirety. In particular, the teachings of all documents herein specifically indicated are incorporated by reference.

1 shows mesenchymal and bone marker expression by bone-forming cells.
2 shows mineralization (A) and ALP (B) staining by bone-forming cells.
FIG. 3 shows ankle diameters (Ankles) in CARRA treated animals (light gray), CARRA + DEX treated animals (dark gray), CARRA + OB treated animals (black) and CARRA + OB + DEX treated animals (dashed lines). Diameter). Diameters are shown as percent increase relative to baseline.
4 shows a comparison between Paw Diameter in CARRA treated animals (diamonds), CARRA + DEX treated animals (squares), and CARRA + OB treated animals (triangles). Diameters are shown as percent increase relative to baseline.
5 shows a comparison between foot diameters in ADJ treated animals (diamonds), ADJ + DEX treated animals (squares), and ADJ + OB treated animals (triangles). Diameters are shown as percent increase relative to baseline.
Abbreviations used in the figures are as follows: CARRA: carrageenan 0.7%; ADJ: 75 μg Complete Freund adjuvant; DEX: dexamethasone 1 mg / kg; OB: osteoblasts 1 x 10 ⁶

Example  One

Experimental procedure

In the following, (A) substantially differentiate the cells of (A) from bone marrow stem cells (BMSC) as described in WO 2007/093431, or (B) in osteogenic medium, or (C) bone from the cavernous bone Procedures are described that result in the induction of bone-forming cells by proliferating blast cells.

A. BMSC From Osteoblasts  Judo

20-60 ml of heparinized bone marrow (BM) was obtained in the iliac crest of patients suffering from bone disease. BM was mixed with phosphate buffered saline (PBS, 2v: v) and layered on density gradient Ficoll solution. After centrifugation, monocytes were harvested from the surface and washed twice with PBS. At the same time, 160 ml of blood drawn in the dry tube was centrifuged to obtain serum from the patient or healthy donor. Cells were resuspended in alpha MEM medium supplemented with 20% allogeneic plasma and 10 ng / ml FGF2 (or another growth factor known in the art to induce osteoblast phenotypes such as BMP). Cells were plated at 1 × 10 ⁷ cells per 175 cm 2 flask and maintained in a 37 ° C. humidified atmosphere containing 5% CO ₂ . The cells were allowed to attach for 4 days before initial medium exchange. Two other partial medium exchanges (half volume exchange) were performed on days 7 and 11. On day 14 cells were separated for 1-5 minutes at 37 ° C. using trypsin-EDTA solution. Cells were counted and plated at 1 × 10 ⁶ cells / 175 cm ² for one more week of culture.

B. Myeloid Mesenchymal Cell Differentiation in Osteogenic Media

Bone marrow mesenchymal stem cells from standard MSC proliferation cultures were incubated with trypsin-EDTA and plated at 60-120,000 cells / well in 6-well plates (12 500 cells / cm 2) in growth medium. The following day, the medium was replaced with 2,5 ml osteogenic medium. Cells were incubated for 2, 3 or 4 weeks. Medium was changed every 3-4 days.

badge

Dexamethasone Diluent:

Dex1 (5.10 ^-4 M): 2 μl dexamethasone stock solution (5.10 ^-2 M) + 198 μl alpha-MEM

Dex2 (10 ^-6 M): 2 μl Dex1 (5.10 ^-4 M) + 998 μl alpha-MEM

Osteogenic  Medium (40 ml)

                 Volume final concentration

Alpha MEM 31 ml /

FCS 6 ml 15%

PenStrepGlu (100x) 400 μl 1x

Dexamethasone (Dex2) 400 μl 10 ^-8 M

200 μl ascorbic acid 50 μg / ml

Beta-glycerophosphate 2 ml 10 mM

C. sponge Osteoblasts  multiplication

The connective tissue and cortical bone were carefully removed from the human bone specimen and the remaining spongy bone was chopped into small sections (1 mm 2). Bone sections were washed extensively with PBS to remove adherent bone marrow cells and seeded in 25 cm 2 tissue culture flasks in culture medium (see above) supplemented with autologous serum with or without growth factors. The medium was changed twice a week. After 4 weeks the cells were released using trypsin-EDTA solution, counted and finally replated to a density of 5000 cells / cm 2.

Flow cell  analysis

The immune-biological cell surface markers of the cells were analyzed by flow cytometry. Bone-forming cells were incubated with the following labeled monoclonal antibodies: HLA-I, HLA-DR, CD80, CD86, CTLA-4, CD40L and CD28 for 15 minutes, then washed with PBS and centrifuged prior to centrifugation and reproduced in 0.3 ml PBS. It was cloudy.

After fixation and permeation of cells OCN was immunodetected with specific antibodies and staining was analyzed by flow cytometry.

Guanghua  black

Osteogenic medium was added to the cells to assess the induction of mineralization potential. About 6 000 bone forming cells (see above) / cm 2 were plated in 6-well plates in the presence of 5% autologous plasma supplemented with 0.1 μM dexamethasone, 0.05 mM ascorbic acid and 3 mM glycerol phosphate. After 2, 3 or 4 weeks of culture, cells were fixed in 3.7% formaldehyde / PBS and stained with alizarin red.

ALP  dyeing

Cells are stained by ALP detection. Bone-forming cells (see above) were washed twice with PBS, then fixed in 60% citrate buffered acetone for 30 seconds at room temperature and then rinsed again with distilled water for 45 seconds. Cells were then stained with Fast Blue RR / Naphtol AS-MX phosphate solution at room temperature for 30 minutes in dark. Cells were washed with distilled water for 2 minutes and then counterstained for 10 minutes in Mayer's Hematoxylin solution. Finally, the cells were washed for 3 minutes with distilled water.

Proliferation assay

200,000 human T cells / ml from individual A (peripheral blood mononuclear cells, PBMCa), together with irradiated or CD3 removed PMBC from individual B (APCb), and human bone-forming cells from a third individual, Plated in a 96-well microtiter plate for 10 days in the presence or absence of PHA (mitogen activator of T cells) in a volume of 200 μl. Human bone-forming cells were seeded at 20,000, 100,000 and 200,000 / ml. When PBMCa and APCb were co-incubated with or without PHA, a mixed lymphocyte response in which PBMCa cells were activated by surface antigens on APCb occurred. Cultures were incubated with 1 μCi / ml 3H-thymidine for an 18 hour incubation period to measure T cell proliferation. Cells were washed twice with ice cooled PBS and twice with ice cooled 5% trichloroacetic acid (TCA). Finally, cells were lysed with 0.1 N NaOH and 0.1% Triton-X100 containing solution. Supernatants were harvested and mixed with the scintillation liquid to be analyzed in a beta-counter.

result

Bone reconstruction characteristics

Levels of cellular markers (bone and mesenchymal markers) or membrane markers were assessed by flow cytometry after 3 weeks (FIG. 1). Bone (ALP, OCN) and mesenchymal (CD105, 73, 90) markers were highly expressed, while hematopoietic markers (CD45) were negative.

Correlation with bone biological function was performed with ALP production (ALP staining) and calcium deposition (mineralization): ALP was expressed on most 21st day by most cells if not all cells and was an important mineral deposit. (Greater than 65% of total area on microscopy) was observed (FIG. 2).

Immune-modulating properties

The results shown in Tables 1 and 2 indicate that APCs are recognized as foreign cells by PBMCs and that PBMCs thus proliferate. When PBMCa and APCb were mixed in the presence of autologous BM derived osteoblasts (from individual B, BMOBb), the mixed lymphocyte response was inhibited by 40-50%.

The same effect was observed in the presence of PHA (10 μg / ml), a potent T cell proliferation stimulant (Table 2). The immunosuppressive effect of osteoblasts was more important (at 55-65%) for stimulated T cells.

Interestingly, when mixed in the presence of allogeneic BM derived osteoblasts (from individual C, BMOBc), the mixed lymphocyte response was also 30-40% in standard conditions (Table 1), 60 in PHA stimulated conditions. Significant inhibition of ˜65%. BM derived osteoblasts (BMOBc) from individual C were loaded into 96-well microtiter plates and co-incubated with PBMCa and APCb. Cultures were incubated with 3H-thymidine for an 18 hour incubation period to measure T cell proliferation. These results suggest that there is no specificity of inhibition for HLA subtypes.

TABLE 1

Table 2: Inhibitory effect of BM derived osteoblasts (BMOB) on PHA activated T cell proliferation (expressed as cpm of incorporated [ ³ H] thymidine)]

conclusion

Bone-forming cells such as bone progenitor cells, pre-osteoblasts or osteoblasts may be useful for treating inflammatory rheumatic diseases because they exhibit both bone reconstruction and anti-inflammatory properties. These cells are characterized by high expression levels of mesenchymal and bone surface markers that correlate with ALP enzyme activity and mineralization ability to corroborate its bone biological profile. The cells can also downregulate the proliferative response of T cells stimulated on an autologous and allogeneic basis. This demonstrated that autologous or allogeneic bone marrow derived bone forming cell products may be particularly useful in the treatment of inflammatory rheumatic diseases.

Example  2: In vivo  Model of inflammation ( IRD )

Carrageenan Induced Ankle / Foot Inflammation Model (1)

A solution containing 1% carrageenan (CARRA; Sigma, Switzerland) in PBS was injected into the hind paw of 8 week old SWISS mice to induce inflammation; Single injection of each hind paw of each animal (Table 3). PBS alone or dexamethasone solution (DEX; Sigma Switzerland) at concentrations of 1 mg / kg, or 1 × 10 ⁶ human bone-forming cells (OB) immediately after injection into carrageenan-injected animals (myeloid mesenchymal as described in Example 1A) Derived from stromal cells) or a combination of DEX and OB.

Table 3: Experimental Protocol

Before injection at TO and T1, T4 and T24 under isoflurane sedation; The circumference of the ankle and foot was measured using digital calipers prior to CARRA administration and at 1, 4, 6 and 24 hours after injection, respectively.

Carrageenan Induced Ankle / Foot Inflammation Models (2)

Inflammation was induced by injecting a solution containing 1% lambda carrageenan (CARRA; Sigma, Switzerland) in PBS into the hind paw of 8 week old SWISS mice; Single injection of each hind paw of each animal (Table 4). PBS alone or dexamethasone solution (DEX; Sigma Switzerland) at concentrations of 1 mg / kg, or 1 x 10 ⁶ human bone-forming cells (OB) immediately after injection into carrageenan injected animals (myeloid mesenchymal phase as described in Example 1A) Derived from tissue cells).

Table 4: Experimental Protocol

Under isoflurane sedation, before injection at TO and T1, T4, T6 and T24; The circumference of the ankle and foot was measured using digital calipers prior to CARRA administration and at 1, 4, 6 and 24 hours after injection, respectively.

Supplements-Induced Ankle / Foot Inflammation Model

Inflammation was induced by injecting a solution containing 0.5% Freund's Complete Adjuvant (ADJ; Sigma, Switzerland) in PBS into the hind paw of 8 week old SWISS mice; Single injection of each hind paw of each animal (Table 5). PBS alone, or dexamethasone solution (DEX) at 1 mg / kg concentration, or 1 × 10 ⁶ human bone-forming cells (OB) immediately after injection into adjuvant-injected animals (myeloid mesenchymal stem cells as described in Example 1A) Derived from).

Table 5: Experimental Protocol

Under isoflurane sedation, before injection at TO and T1, T4, T6 and T24; The circumference of the ankle and foot was measured using digital calipers before ADJ administration and 1, 4, 6 and 24 hours after injection, respectively.

result: Carrageenan Effects on induced inflammation

Carrageenan injection induced an immediate inflammatory response that was measurable by increasing the diameter (foot or ankle) in the injected animals. The increase in diameter beyond baseline was approximately 20% after 1 hour, 25% after 4 hours, and 17% after 24 hours (FIG. 3).

At the ankle, dexamethasone (1 mg / kg) induced strong inhibition of inflammation and swelling of the foot / ankle injected with carrageenan (FIG. 3). Inhibition of inflammation induced by dexamethasone was 100% at 1 and 4 hours, but then began to deviate and was completely lost at 24 hours.

Compared to the ankle, administration of osteoblasts induces moderate but significant inhibition of inflammation at 30% and 40% reduction at 1 and 4 hours, respectively, but the inhibition lasts as long as the inhibition is maintained at 40% at 24 hours. It seems to last.

Interestingly, a synergistic (and potent) effect of the combination of bone forming cells and dexamethasone was observed in carrageenan-induced inflammation (FIG. 3). Anti-inflammatory effects were 50%, 80% and 75% at 1, 4 and 24 hours, respectively.

The anti-inflammatory effect on bone-forming cells was stronger in the paws and lasted longer. Dexamethasone showed 100%, 70% and 38% inhibition at 1 h, 6 h and 24 h, respectively, while osteoblasts showed 80%, 90% and 95% inhibition (FIG. 4). This may be due to better distribution and spread of the injected cells of the foot.

Results: Effect on Adjuvant-Induced Inflammation

In adjuvant-induced inflammation, despite the more severe inflammation of the foot / ankle (ie 40-50% in diameter increase from baseline at 4-6 hours), osteogenic cells are similar to dexamethasone inhibition maintained at 24 hours. It showed a tendency to exhibit a strong anti-inflammatory effect (peak effect of 75-90%) (FIG. 5).

Claims (10)

Isolated bone-forming cells for use in treating the inflammatory component of inflammatory rheumatic disease (IRD). The isolated bone-forming cell of claim 1, wherein the bone-forming cell may be a) undergoing osteogenic differentiation, or b) is involved in osteogenic differentiation, or c) at least partially progresses along osteogenic differentiation. The isolated bone-forming cell of claim 1 or 2, wherein the bone-forming cell is characterized by:
i) the cells comprise alkaline phosphatase (ALP), more particularly expression of ALP of bone-liver-kidney type, and / or expression of osteocalcin (OCN);
ii) show evidence of the ability of cells to mineralize the external environment or to synthesize calcium-containing extracellular matrix; And
iii) the cell is substantially not differentiated into any of the cells of the adipocyte lineage or the chondrocyte lineage, preferably neither. The isolated bone-forming cell of claim 1, wherein the bone-forming cell is of human origin and the medicament is used for autologous or allogeneic administration to a human subject. The isolated bone-forming cell of claim 4 wherein said administration is systemic, topical, intraarticular or perarticular administration. 5. The method of claim 1, wherein the IRD comprises osteoarthritis (OA), psoriatic arthrosis, gout, pseudogout, and rheumatoid arthritis (RA), enteroarthritis, reactive arthritis, and lighter syndrome. Arthritis of various origins, osteonecrosis, minor joint juvenile rheumatoid arthritis, Still's disease, Behcet's disease, systemic lupus erythematosus, septic arthritis, and spondyloarthropathies including ankylosing spondylitis, enteropathic spondylitis and undifferentiated spondyloarthropathy Isolated bone-forming cells. 5. The isolated bone-forming cell of claim 1, wherein the IRD is other than osteoarthritis (OA), rheumatoid arthritis (RA), and osteonecrosis. The isolated bone-forming cell of claim 1, wherein the IRD comprises both inflammation and bone lesion (s). 8. The isolated bone-forming cell of claim 1, wherein the IRD comprises inflammation but does not comprise bone lesion (s). Selected from bone-forming cells, and disease-modulating antirheumatic drugs (DMARDs), glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs) and analgesics for simultaneous, sequential or individual use in the treatment of inflammatory components of IRD Pharmaceutical compositions comprising a formulation.

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